A chlorine amount denotes a hypochlorous acid, which is generated when a chlorine agent is dissolved in water and a chloroamine, which is generated when this is connected with ammonia. The former is called a free chlorine and the latter a combined chlorine. For the method of determining these chlorine amount, for example, o-tolidine colorimetric method, or is usually used. Besides these, when the chlorine amount is relatively high, iodine titration is also applied.
In the present invention, the total chlorine amount means a sum of the free chlorine amount and the combined chlorine amount.
Benzidines such as TMB and SA-TMB is a coloring substrate frequently used for determining peroxidase activity in enzyme-linked immunosorbent assay (ELISA) (for example, see Tetrahedron, volume 30, pp. 3299-3302 by Holland, and Japanese Patent O.P.I. Publication No. 52300/1986). In ELISA, in order to measure fine amounts of material existing in vivo, the following theory is commonly utilized: a binder material such as an antibody against aforesaid material is fixed on a cavity (well) of beads or special plates which are called "carriers", and then, a solution containing aforesaid material was reacted for binding. Following this, a second material (for example, an antibody) possessing binding property to aforesaid material which has been peroxidase-labeled is allowed to react and bind. Next, the TMB was added to aforesaid material under the existence of hydrogen peroxide. As a result, due to the effect of the peroxidase, hydrogen peroxide generates oxygen. As a result, the TMB is oxidized so as to make coloring. Due to this theory, the correlation between the degree of light absorbance of the coloring material and aforesaid material concentration can be utilized.
Namely, benzidine compounds such as TMB and SA-TMB are used for measuring hydrogen peroxide under the existence of peroxidase.
In accordance with JIS K0102, an o-tolidine or a DPD colorimetric method determines chlorine amount in a solution by comparing a yellow coloring of potassium chromate--potassium dichromate solution or a pink coloring of disodium 1-(4-methylbenzenesulfonamide)-7-(2-methylphenylazo)-8-hydroxy-3,6-naphtha lene sulfonic acid chlorine standard solution with a dye generated due to mixture of the solution to be tested and o-tolidine or DPD.
According to this method, it is possible to precisely determine the chlorine amount. However, chlorine standard calorimetric solutions having various concentrations (for example, 20 sample tubes in which a can concentration is increased from 0.01 mg/l to 0.2 mg/l at an increment of 0.01 mg/l.) must be prepared. Since this standard solution cannot be free from color-fading, it is impossible to store for a long time. Therefore, it is necessary to be prepared when it is necessary. In addition, when precipitation occurs when preparing, preparing must be conducted again. Accordingly, its procedure is very complicated.
On the other hand, for measuring chlorine amount having relatively high concentration, an iodine titration method is used. According to JIS K 0102, in this method too, a potassium iodide solution and the solution to be tested for chlorine are mixed so that isolated iodine is titrated with sodium thiosulfate. After yellow color of the solution is thinned, a starch solution is added as an indicator. The mixed solution is titrated until blue color of starch is extinguished. By the use of the amount of sodium thiosulfate required for titration, chlorine amount can be calculated from a certain equation. However, this method is also very complicated in terms of necessitating titration operation.
Cases in which measurement of chlorine is required can be considerable in various ways. For example, in food manufacture, when a hypochlorous acid solution is used for sterilizing of pipes, tanks, etc., in the manufacturing steps of liquid foods, it is necessary for the hypochlorous acid solution to be removed by washing. However, in the process control of routine steps like this, it is extremely difficult to undergo complicated and elaborate inspections, and, as a result, it is possible for such determinations to be left to the intuition of those skilled in the art.
Concerning coloring of the TMB, Japanese Patent Examined Publication No. 25152(1990) discloses that two sequential coloring steps takes place. The two sequential coloring steps explained therein is assumed to be the first kind in blue, and, next, in the second kind in brown. This first coloring in blue tends to be only temporary, and finally changes into brown, That is to say, this indicates that blue color subsequently changes into brown with lapse of time. This reference also discloses that this phenomenon occurs more easily with higher analytical densities. However, the above-mentioned reference only refers to the existence of the first and second kinds (i.e., blue and brown colors) and the inventors attention in this patent is focused on only how to maintain the blue color, and, as regards the brown dye which subsequently appears in time, it only refers to it as being an unpreferable phenomenon. Thus, in this respect the reference does not disclose the relationship of the color hues, which change depending upon the mol ratio between the TMB and the oxidant.
In the above-mentioned o-tolidine colorimetric method, the reaction solution appears yellowish, therefore, the change of the yellow density is used only for determination of chlorine, and change of hue was not at all utilized. In addition, in accordance with Holland's method, the reacted solution appears bluish, therefore, only change of blue-density was used for determination of peroxidase activity. In Holland's method, change of hue was not utilized at all.
Accordingly, the reference neither discloses nor indicates the color changes stably in blue, green, yellow, orange or red depending on the concentration of the oxidant.
Further in TMB, when the concentration of TMB is high, there is a problem that dye (this phenomenon remarkably occurs in the case of blue color) after coloring, tends to precipitate when it is stored for a pre-determined period of time. In Japanese Patent O.P.I. Publication No. 62-182,659(1987), it is disclosed that by making active use of the nature of certain kinds of dyes caused by specific kinds of anion compounds, said insoluble dye is used for visualizing biological substances such as dyeing of immuno-tissues, etc.
The first object of the present invention is to provide a method of determining total chlorine amount in a test solution by the use of a phenomenon in which said total chlorine reacts with benzidine compound so as to form a dye, and the formed dye changes in the hue depending upon a mol ratio of the total chlorine amount to the benzidine compound and a kit used for determining total chlorine amount, and the above-method can be carried out by simple operation and simply by observing the hue of the generated color in the test solution.
Further, the second object of the present invention is to provide a method of determining total chlorine amount, which is capable of restraining precipitation of dyes even if coexisting a benzidine compound with a chlorine, and a kit used therefore.